Reaction Protocol for EpiMark® 5-hmC and 5-mC Analysis Kit (E3317)


  1. DNA Glucosylation and Control Reactions

    1. Mix the following components in a 1.5 ml reaction tube:

      Genomic DNA 5–10 μg 30 μg/ml
      UDP-Glucose 12.4 μl 80 μM
      NEBuffer 4 31 μl 1X
      Nuclease-free water to 310 μl Total vol. 310 μl
    2. Split the reaction mixture into two tubes (155 μl each).

    3. Add 30 units (3 μl) of T4 β-glucosyltransferase (T4-BGT) to one tube. Mix well by pipetting gently up and down. (The second tube is the control reaction. Add 3 μl of nuclease-free water).

    4. Incubate both tubes at 37°C for 12 to 18 hours.

  2. Restriction endonuclease digestion

    1. Aliquot 50 μl of each reaction mixture into three 0.2 ml PCR-strip tubes. Label tubes 1–3. Repeat for control experiment. Label tubes 4–6.

    2. Add 100 units (1 μl) of MspI into tubes #1 and 4. Mix well by gently pipetting up and down.

    3. Add 50 units (1 μl) of HpaII into tubes #2 and 5. Mix well by gently pipetting up and down. (tubes #3 and 6 are controls, no restriction enzyme is added).

      DNA + T4-BGT+ UDP-Glc DNA + UDP-Glc (Control)
      1 2 3 4 5 6
      MspI HpaII Control (no RE) MspI HpaII Control (no RE)
    4. Incubate the reactions at 37°C for at least 4–16 hours.

    5. Add 1 μl Proteinase K, Molecular Biology Grade to each tube and incubate at 40°C for 30 minutes. Inactivate Proteinase K by incubating at 95°C for 10 minutes.

  3. Analyze DNA by PCR/qPCR
    End-point PCR: Protocol is provided for NEB LongAmp™ Taq, which has been shown to perform well. Other PCR protocols can be substituted.
    1. Add the following components to a 0.2 ml PCR reaction tube on ice:
      5X LongAmp Taq Reaction Buffer 10 μl 1X
      10 mM dNTPs 1.5 μl 300 μM
      10 μM Forward Primer 1 μl 0.2 μM
      10 μM Reverse Primer 1 μl 0.2 μM
      Template DNA (from Step II) 3 μl 50–100 ng
      LongAmp TaqDNA Polymerase 1 μl 5 Units/50 μl PCR
      Nuclease-free Water To 50 μl  
    2. Gently mix the reaction. If necessary, collect all liquid to the bottom of the tube by a quick spin. Overlay the sample with mineral oil if using a thermocycler without a heated lid.
    3. Transfer PCR tubes from ice to a thermocycler with the block preheated to 94°C and start the cycling program.
      Thermocycling conditions for a routine 3-step PCR:
      Initial Denaturation 1 94°C 30 Seconds
      Denaturation 30 94°C 15 Seconds
      Annealing 55–65°C 30 Seconds
      Extension 65°C 20 Seconds (or 50 Seconds/kb)
      Final Extension 1 65°C 5 Minutes
  4. Real time PCR: 
    For Real Time PCR use 1–2 μl (30–60 ng) of template (from Step II) and follow the manufacturer’s recommendations. 

    Data described in this manual was generated using the DyNAmo™ SYBR Green qPCR Kit with the Bio-Rad iQ™ 5 Real-time PCR Detection System.
    PCR Amplification 95°C 10 min x 1
    95°C 10 sec x 40
    60°C 30 sec
    72°C 20 sec
    Melting Curve 65°C and increment of 0.5°C per cycle 20 min 1

    If using a standard curve to determine copy number, samples can be normalized by dividing the copy number of reactions 1–5 by the copy number of the control reaction (tube 6). If using the comparative Ct method, samples can be normalized by setting the control reaction (tube 6) as the calibrator. This normalization will give an approximate percentage of methylated (HpaII digested samples, (tubes 2 & 5) and hydroxymethylated (T4-BGT & MspI digested sample, tube 1) alleles in your sample.